Identification and evaluation of probiotic potential of Bifidobacterium breve AHC3 isolated from chicken intestines and its effect on necrotizing enterocolitis (NEC) in newborn SD rats

Necrotizing enterocolitis (NEC) is a severe intestinal disease of the newborn infants, associated with high morbidity and mortality. It has been reported that Bifidobacterium could protect the intestinal barrier function and reduce the risk of NEC. This study aimed to evaluate the probiotic potential of Bifidobacterium strains isolated from the chicken intestines and its effect on necrotizing enterocolitis in newborn SD rats. Out of 32 isolates, B. breve AHC3 not only exhibited excellent probiotic potential, including tolerance to artificial simulated gastric conditions, adhesion to HT-29 cells, antioxidant capacity and antibacterial activity, but also possessed reliable safety. Additionally, NEC model was established to further investigate the effect of B. breve AHC3 on necrotizing enterocolitis in newborn SD rats. It was illustrated that administration of B. breve AHC3 significantly not only reduced the incidence of NEC (from 81.25% to 34.38%) (P< 0.05), but also alleviated the severity of ileal injury (P< 0.05). Compared with NEC model, B. breve AHC3 could significantly decrease the level of proinflammatory factor TNF-α (P< 0.05) and increase the level of antiinflammatory factor IL-10 (P< 0.05) in the ileum of NEC rats. Through the intervention of B. breve AHC3, the gray value of inducible nitric oxide synthase (iNOS) in intestinal tissue of NEC rats was significantly reduced (P< 0.05). It was indicated that B. breve AHC3 exhibited prominent probiotic potential and reliable safety. In the neonatal SD rat model of NEC, B. breve AHC3 had an available protective effect on the intestinal injury of NEC, which might be related to reducing the inflammatory reaction in the ileum and inhibiting the expression of iNOS in intestinal tissue cells. B. breve AHC3 could be used as a potential treatment for human NEC.


Introduction
Necrotizing enterocolitis is the most common and serious intestinal disease with fatal risk in newborns, especially premature infants, which is seriously harmful to the health and life of newborns [1,2].NEC usually represents symptoms of anorexia, weight loss, abdominal distension and hematochezia in clinic, and the main pathological manifestation of NEC is inflammatory hemorrhagic necrosis of intestines [3,4].It is reported that the number of newborns diagnosed with NEC accounts for 2%-5% of the total number of newborns in the intensive care unit, the mortality rate is 15%-30% of the confirmed cases, and the mortality rate of newborns requiring surgical intervention is even higher [5][6][7].In addition, the inevitable complications, including short bowel syndrome, heterotopic disease and intestinal stenosis, seriously interfere with the quality of life of infant suffering from NEC [8,9].The pathogenesis of NEC is complex and multifactorial [10].Although decades of research have been carried out on NEC, its pathogenesis is still unclear, and there is still lacking of effective prevention and treatment measures.Evidence suggests that the main risk factors resulting in NEC consist of premature delivery, enteral nutrition, pathogenic bacterial colonization, hypoxia and intestinal ischemia [1,11].
It has been proved that risk factors of NEC could induce inflammatory cascade reaction [12].Excessive intestinal inflammation plays an important part in the pathogenesis of NEC.Cytokine is the key regulator of inflammation during NEC [13].The severity of cytokine production is considered as a biomarker of intestinal injury.Tumor necrosis factor-α (TNF-α) and Interleukin-10 (IL-10) are regard as a crucial diagnostic value in NEC [14].TNF-α, as a significant proinflammatory cytokine, is closely related to intestinal inflammation and NEC [15].A series of cytokines such as interleukin-1 (IL-1), IL-6, IL-8, etc. produced through cascade reaction are caused by TNF-α, the transcription and expression of inflammatory genes are triggered, leading to excessive inflammatory cascade reaction [9,16].Subsequently, the increase of TNF-α level aggravates the intestinal mucosal injury [17].It has been confirmed by experiment that the level of TNF-α in infant suffering from NEC increases [18].IL-10, belonging to the kind of antiinflammatory cytokine, is initially described as an inhibitor on cytokine secretion, which probably has a protective effect on NEC by inhibiting inflammatory reaction.IL-10 plays an antiinflammatory part in the immune system to place restriction on the extent of immune response [19].Study shows that spontaneous enterocolitis occurs in mice lacking of IL-10 [20].On the contrary, application of IL-10 can reduce inflammatory reaction in a mouse model of intestinal ischemia/reperfusion [21].The inflammatory cascade reaction is triggered by risk factors of NEC, which in turn results in the excessive production of iNOS [22].It has been suggested that acute NEC in newborn is in connection with increased expression of iNOS and nitrosation [23].Therefore, searching for new effective drugs to help treating and preventing the development of NEC will become a more popular trend at present.
Probiotic is defined as a group of living and non-pathogenic microorganisms colonizing in the intestine and providing benefits to the host [24].Bifidobacterium is one of the most commonly used probiotic in clinical treatment presently [25].Bifidobacterium is Gram-positive, anaerobic, polymorphous, non-motile and non-spore forming, which includes more than 30 species currently [26].It is dominant in the intestinal flora of normal newborns [27].Bifidobacterium is beneficial to many aspects of human health through different ways, mainly including enhancement of intestinal barrier, regulation of immune system, inhibition on pathogens, nutrition supplement as well as reinforcement and expansion of host metabolism [28,29].Specific probiotics, especially Bifidobacterium, have been proved to protect intestinal integrity, maintain intestinal barrier function and have beneficial effects on NEC [13,30].Khailova et al. found that oral treatment of B.bifidum could prevent the intestine from disease in the NEC model [13].Underwood et al. observed that administration of B. longum subsp.infantis had protective effect on NEC and reduced NEC-related inflammation [31].It was documented by Wu et al. that B. adolescentis significantly reduced the incidence of intestinal injury and prevented the morbidity of NEC in premature newborn rats [32].Therefore, biological exploration of new strains with probiotic potential, especially beneficial effect on NEC, has become a hot spot of current research on prevention and treatment of NEC.
The chicken gastrointestinal microflora has attracted more and more attention in recent years due to its biodiversity [33].The avian gastrointestinal microflora is composed of tens of billions of microbes, which contributes to digestion and absorption of nutrients, immune system development and pathogen inhibition [34].It plays an important role in maintaining the normal and harmonious functions in the whole organism.In the chicken gastrointestinal microflora, approximately 900 species of microbes identified belong to 117 genera [35].It has been reported that the content of Bifidobacterium in poultry cecum is up to 10 9 −10 10 CFU/g [36].Therefore, the chicken gastrointestinal tract is regarded as an excellent area for screening more strains with significant probiotic characteristics.Aalipanah et al. reported that a strain of B.bifidum isolated from the chicken intestine showed potential probiotic characterization [37].A strain of B. dentium from chicken intestines was isolated by Palacios et al., exhibiting the highest phytase activity [38].Collado et al. also isolated 4 B. longum strains from chicken intestines, it was indicated that the adhesion of Bifidobacterium strains originated from animal was significantly better than that of Bifidobacterium strains from human [39].
Hefei free-ranged chicken is an excellent local traditional breed of chicken originating from the central and western regions of Anhui, China.It belongs to the flaxenfeather chicken and has a long growth period.The chicken mainly lives in hilly areas and is limited to scattered breeding by local farmers currently, which has strong foraging ability and mainly feeds on vegetable leaves, grass seeds, leaves, and small insects.However, it has so far been rare reports on the isolation, identification, and probiotic potential of Bifidobacteria in the gastrointestinal tract of Hefei free-ranged chicken, in despite of a significant increase in literature on the screening and probiotic potential of probiotics in the chicken gastrointestinal tract in the past decade.
The purpose of the present study was two folds: first, to evaluate the probiotic potential of Bifidobacterium isolated from the free-ranged chicken intestines in vitro, mainly including tolerance to artificial simulated gastrointestinal conditions, adhesion ability to HT-29 cells, antioxidant capacity and antibacterial activity.Second, a NEC model was established by hypoxia, cold stimulation, artificial feeding and LPS induction to further investigate the effect of B. breve AHC3 on necrotizing enterocolitis in newborn SD rats in vivo, through observing incidence rate of NEC, severity of ileal injury, level of inflammatory factors and expression of iNOS, to provide theoretical basis for clinical application of B. breve AHC3 to prevent and treat NEC.

Isolation, purification and preliminary identification of Bifidobacterium spp.
The ilea and caeca of 10 healthy adult free-ranged chickens (Gallus gallus) were collected from peasant households in Hefei(E 116˚42 0 19 00 , N 31˚46 0 25 00 ), Anhui Province, China and immediately transported to the laboratory under sterile and coolingconditions.The intestineswere cut open with sterile scissors, the inner wall of the intestine was rinsed with sterile normal saline to remove the contents of the intestine.Onegramof intestinal mucosa was scraped and put into 9 mLPBS containing 0.05% L-cysteine hydrochloride (Sigma Aldrich, USA)(PBSc) for tenfold serial dilutions, subsequently.A volume of 0.1mL sample solutionwasspreadon selective de Man, Rogosa, and Sharpe (MRS, HopeBio, China) platescontaining 0.05% (w/v) L-cysteine hydrochloride(MRSc) plus 100 mg/L mupirocin (HopeBio, China), and placed in anaerobic environment for culture at 37˚C for 72 h.Selection and purification the single white colonies by streaking on selective MRSc plate.Observationon colony morphological characteristics, Gram staining,catalase production and biochemical experiments were included in the preliminary identification on isolates.All the experiments in this study have been carried out in triplicates.The commercial strain Bifidobacterium animal subsp.Lactis BB-12 (BB-12) with good probiotic characteristics was selected as the reference strain, which was provided by the Institute of Microbiology, Anhui Academy of Medical Sciences, China.

Evaluation of probiotic potential of Bifidobacterium spp.
Tolerance to artificial simulated gastrointestinal conditions.According to the method described previously, tolerance to simulated gastrointestinal conditionsof isolated Bifidobacterium strains was determined with simple modifications [40].The isolated strains were anaerobically cultured in MRSc broth supplemented with 0.1% (w/v) ascorbic acid at 37˚C for 48 h, centrifugation by 10000 × g for 10 min at 4˚C and the particles were washed twice with PBSc buffer (pH = 7.4).Using 4 mL of artificial simulated gastric juice (125 mM NaCl, 7 mM KCl, 45 mM NaHCO 3 , 3 g/L pepsin (Shanghai Lanji Technology Development Co., Ltd.), pH = 2.0 and sterilization by filtration) to suspend the particles (10 8 CFU/mL), fowlled byincubation in 37˚C water bath for 3 h.After tenfold serial dilutions, 0.1 mL suspension was spread onMRScplate, and the number of viable cells was measured by colony count.After treatment with artificial simulated gastric juice for 3 h, the isolateswere obtained under centrifugation by 10000 × g for 10 min at 4˚C and the particles were washed twice with PBSc buffer.Using 4 mL of artificial simulated intestinal juice (22 mM NaCl, 3.2 mMKCl, 7.6 mM NaHCO 3 , 1 g/L trypsin (Shanghai Lanji Technology Development Co., Ltd.), 0.3 g/L bile oxgall (Beijing Solarbio Science and Technology Co., Ltd.), pH = 8.0 and sterilization by filtration) to suspend the particles, fowlled by incubation in 37˚C water bath for 5 h.After tenfold serial dilutions, 0.1 mL suspension was spread on MRSc plate, and the number of viable cells was measured by colony count.
Adhesionto HT-29 cells.Adhesion of isolated Bifidobacterium strains to HT-29 cells was evaluated as the method described by Lee et al. [41] with slightmodifications.HT-29 cells (Hunan Fenghui Biotechnology Co., Ltd.) were cultivated in Dolbecco'sModified Eagle Medium F-12 (DMEM/F12, Gibco, USA)culture supplemented with 10% (v/v) fetal bovine serum (ZhengjiangTianhang Biotechnology Co., Ltd.) in a 5% CO 2 incubator at 37˚C for 48 h.The cells were harvested and added into sterile 24-well plate (10 6 cells/well), followed by cultivation for 48 h.The culture was refreshed every 24 h.Added 0.5 mL of Bifidobacterium strains (10 8 CFU/mL) into the wells and incubated the mixture at37˚C for 2 h anaerobically.The wells were washedfor 3 times with PBSc solution, then a drop of Triton X-100 (Biofoxx, Germany) was added into each well, and the lysates were subsequently spread on MRSc plates.The number of Bifidobacterium was measured by colony count.
Antioxidant capacity.The radical scavenging activity of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was detected using the procedure [42] with minor modifications.In brief, 0.2 mLPBS containing 10 9 CFU/mL Bifidobacterium was added into 1 mL DPPH methanol solution (100 μM).After exposed in the dark at 37˚C for 20 min, the mixturewas centrifugated at 8000 × g for 5 min and the absorbance value at 517 nm was measured.According to the methodology used [40], the radical scavenging activity of 2, 2-azinodi-[3-ethylbenzthiazoline sulfonate] (ABTS) was investigatedwith simple modifications.A volume of 0.1 mL PBS containing 10 9 CFU/mL Bifidobacterium was added into 1 mLABTS working solution (7 mM ABTS solution, 2.45 mM potassium persulfate solution and incubation in the dark at 37˚C for 12 h).After exposed in the dark at 37˚C for 20 min, the mixture was centrifugated at 8000 × g for 5 min and the absorbance value at 734 nm was measured.The radical scavenging activity of superoxide anion was assayedas the method described previously [43] with slight modifications.A volume of 0.5 mL PBS containing 10 9 CFU/mL Bifidobacterium was added into 1.5 mL Tris-HCl solution (pH = 8).After placed at 25˚C for 20 min, 0.2 mL of pyrogallol solution (25 mM) was added into the mixture.After exposed in the dark for 5 min, the reaction was terminated with 0.25 mL hydrochloric acid.The mixture was centrifugated at 8000 × g for 5 min and the absorbance value at 325 nm was measured.
Antibacterial activity.Antibacterial activity of Bifidobacterium strains was performedwith the protocol described by Piyadeatsoontorn et al. [44] with some modifications.In this research, Escherichia coli ATCC8099, Staphylococcus aureus ATCC6538, Salmonella enterica ATCC9120 and Shigellasonnei BNCC192105 were applied as indicator strains.First, 0.1mL fresh indicator bacteria culture (10 7 CFU/mL) was spread on LB plate.After the plate surface was air-dryin super-clean worktable, the sterile discswere placed on the plate.A drop of cell free supernatant (CFS) of Bifidobacterium was added onto the disc, and the plates were incubated at 37˚C for 24 h.The diameter of inhibition circle was measured byvernier caliper to determine the antibacterial activity of Bifidobacterium strains.

Evaluation of safety
Hemolysis test.Hemolysis test was evaluated according to the method detailed by Menezes et al. [45].Bifidobacterium strain was streaked on Columbia agar plate containing 5% sheep blood and the hemolytic result was observed after incubation for 24 h.Presence of hydrolytic circle (β-hemolysis) was classified as a positive result.Presence of green circle (αhemolysis) or no hydrolytic circle (γ-hemolysis) was classified as non-hemolysis.
Antibiotic sensitivity assay.Antibiotic sensitivity was measured by the method used [46] with slight modifications.A volume of 0.1 mL fresh Bifidobacterium culture (10 7 CFU/mL) was spread on MRSc plate.After the plate surface was air-dryin super-clean worktable, the antibiotic discswere placed on the plate with sterile tweezer, and the plates were incubated at 37˚C for 24 h.The diameter of the inhibition zone was measured with a vernier caliper to determine its antibiotic sensitivity, and the results were classified according to the microbiological breakpoints for antimicrobials issued [47].
Biogenic amines production.The production of biogenic amines was detected as protocol described previously [49].Pyridoxal-5-phosphate (0.005%) as a cofactor in the decarboxylation reaction was added to the MRSc medium (pH = 5.3).The Bifidobacterium strain was streaked on the MRSc plate supplemented with amino acids (lysine, histidine, arginine, tyrosine and ornithine) (Beijing Solarbio Science and Technology Co., Ltd.), and the final concentration was 0.5%.Subsequently, the plate was incubated at 37˚C for 72 h.The positive result was confirmed through the color changed from yellow to purple of the indicator bromocresol violet.16s rRNA gene sequencing for molecular identification.The isolated Bifidobacterium strain was identified by 16S rRNA gene at the species level.Genomic DNA was extracted and used for PCR amplification [44].Briefly, the 16S rRNA gene was amplified by using the primer pair 27f (5 '-AGAGTTTGATCCTGGCTCAG -3') and 1492r (5 '-CTACGGCTA CCTTGTTACGA -3').Total 50μL system as follows: 1.0μL template DNA, 1.0μLTaq DNA polymer, 5.0μL 10 × PCR buffer, 1.0μL10 mM dNTP, 1.5μL 10μM upstream and downstream primers,respectively, 39.0μL ddH 2 O, andthermal cycle parameters performed as follows: initial denaturation at 95˚C for 300 s, 35 denaturation cycles at 95˚C for 30 s, annealing at 58˚C for 30 s, extension at 72˚C for 90 s, and final extension at 72˚C for 420 s for PCR reaction.A volume of 3 μL PCR amplification fragment was visualized at100 V for1 h by using 1% agarose for electrophoresis.The PCR product was purified and sequenced by Shanghai personal Gene Technology Co., Ltd.16S rRNA gene sequences were compared and matched with available sequences in NCBI GenBank using BLAST.The homology of the target gene sequence was analyzed, and the phylogenetic tree was constructed with MEGA 7.0, based on the bootstrap value of 1000 replications.The identified sequence was uploaded to NCBI gene bank database.
Scanning electron microscope (SEM) observation.In order to observe the morphology of isolated Bifidobacterium strain exhibiting probiotic potential, the strain was incubated at 37˚C for 18 h in MRSc broth, and the culture was observed under a scanning electron microscope by using critical point drying (CPD) method [50].
Ethics statement.The protocol was approved by the experimental animal ethics committee of Anhui Academy of Medical Sciences.According to the procedures approved by the Institutional Animal Care and Use (IACUC), euthanasia must be carried out in rats when the animal meets one of the following criteria: (1) loss of appetite and significant weight loss, (2) sharp decrease in activity, loss of sensitivity to external stimuli, pale and cyanotic skin, and no response to treatment, (3) poor prognosis and moribund state.Daily inspections were conducted by professionals to assess the health status of rats.At the end of the experiment or one of the above criteria, euthanasia to rats were carried out by decapition to alleviate the pain of the animals, and the number of rats used was minimized as much as possible based on the completion of the experiment.Throughout the modeling process, no surgical procedure was performed.After conducting hypoxic cold stress experiments, timely oxygenation treatment and rapid warming to rats were carried out to reduce discomfort in the animals.In addition, the rats were treated as gentle as possible during the experimental operation and comforted as much as possible after the experiment.
Establishment of the NEC model.128 newborn Sprague-Dawley rats without specific pathogen within 2 h after birth were purchased from Laifu Animal Breeding Farm, Nanjing, China(SPF grade).128 SD rats were randomly divided into 4 groups.Group A was the blank control group (n = 32), Group B was Bifidobacterium control group (n = 32); Group C was NEC model group (n = 32), and group D was Bifidobacterium intervention group (n = 32).Group A and B were fed by female rats, Group C and D were fed with artificial formula milk powder every 4 h.The rats were given 0.1 mL by gavage for the first time, 0.1mL increased the next day, and the maximum amount was no more than 0.3 mL.The NEC model of neonatal SD rats was established by hypoxia-cold stress, artificial formula feeding and LPS (Sigma, USA) treatment according to the method as previouslydescribed by Xiang [51] with slight modifications.Group C and D were treated with hypoxic-reoxygenation (first breathing 100% nitrogen for 3 min, then immediately breathing 100% oxygen for 3min), and a cold stress (4˚C, 10 min) twice a day for 3 d.Meanwhile, neonatal SD rats were induced NEC by intraperitoneal injection of LPS (5 mg/kg, prepared with normal saline to 5 mg/mL) once a day for 3 d.Group B and D were treated by Bifidobacterium gavage after daily modeling stimulation, and Bifidobacterium was added to 10 mL normal saline or artificial formula milk (5 × 10 9 CFU/ mL), 0.2 mL a day by gavage for 3 d.Group A and C were treated with the same amount of normal saline instead.Group A and B were not treated with modeling stimulation.
Pathological observation on intestinal tissue.A 1 cm of the proximal intestinal tube of ileum was taken out aseptically and fixed in 10% formaldehyde solution, embedded in paraffin, coronal sectioned, and stained with hematoxylin eosin (H & E) to observe the pathological change of the intestine under light microscope.According to the pathological scoring criteria reported [52], the intestinal tissue damage was observed and scored by the blind assessor.It was divided into 4 grades as follows: 0, normal; 1 point, slight separation of submucosa and / or lamina propria; 2 points, moderate separation of submucosa and / or lamina propria, edema of submucosa and/or muscle layer; 3 points: severe separation of submucosa and / or lamina propria, severe edema of submucosa and / or muscle layer, local villi shedding; 4 points: intestinal villi disappeared with intestinal necrosis.Histological score�2 indicated NEC positive.
Determination of TNF-α and IL-10 levels.The levels of TNF-α and IL-10 in intestinal tissue supernatant were investigated by Enzyme-linked immunosorbent assays (ELISA).In short, the intestinal tissue was collected and dissected, the contents of the intestinal cavity were washed with ice physiological saline.After dried with filter paper, the intestinal tissue was homogenized with a homogenizer, followed by centrifugation at 4˚C and 10000 r/min for 15 min.Rat TNF-α ELISA detection kit (Shanghai Hufeng Chemical Co., Ltd.) and rat IL-10 ELISA detection kit (Shanghai Hufeng Chemical Co., Ltd.) were used to measure the levels of TNF-α and IL-10 in intestinal tissue according to the manufacturer's instructions.
Detection of iNOS in intestinal tissue.The determination of iNOS in intestinal tissue was carried out by immunohistochemical staining as the methodologyused by Underwood [53].The terminal ileum tissue was fixed and sectioned conventionally.After deparaffinization and rehydration, the sections were repaired with citric acid buffer (pH = 6.0), and then incubated with 50 μL 0.3% hydrogen peroxide for 10 min at room temperature to block the activity of endogenous peroxidase.The sections were incubated sequentially with 50 μLiNOSprimary antibody for 60 min, 50 μL polymer enhancer (reagent A) for 20 min and 50 μL enzyme labeled anti-rat polymer (reagent B) for 30 min at room temperature.The sections were washed 3 times by PBS after each incubation, followed by visualization with diaminobenzidine (DAB) and hematoxylin counterstaining, respectively.Finally, the sections were sealed.PBS was used as a negative control instead of primary antibody and the specimens were observed under optical microscope.Five different visual fields were randomly selected for observation in each specimen.In the visual field, tan sediment was declared to be a positive reaction, and its density and positive expression site represented the number of antigen distribution and the location of antigen.The average positive staining gray value of iNOS was obtained by image analysis software (Image Pro Plus).The gray value ranged from 0 to 255, and the darker the color of sediment, the larger the measured value.
Statistical analysis.Experiments were performed in triplicate in the present research,and results were presentedas mean ± SD.SPSS version 23.0 was employed for data analysis.The variance and differences wereanalyzed by One-way ANOVA with Duncan's multiple range test and P values < 0.05 were considered statistically significant.

Preliminary identification on isolated strains
In the present work, a total of 32 strains were isolated from 10 chicken intestine samples, preliminary physiological and biochemical identification were carried out for 32 isolates by microbiological and biochemical methods.With reference to the physiological and biochemical identification results of Bifidobacterium in Berger's Manual for identification of bacteria, as well as the classification and identification methods for Bifidobacterium, 21 isolated strains were preliminarily identified as Bifidobacterium spp.(Table 1).All 21 isolates were Gram-positive, and Gram staining morphology of representative strain AHC3 was short rod or ellipsoid shape, appearing in pairs or clusters.Colony morphology of AHC3 was medium-sized or smaller, white or milky white, round, moist, soft, opaque, and neat edges.Colony morphology and Gram staining morphology of representative strain AHC3 were shown in Fig 1A and 1B.

Tolerance to artificial simulated gastrointestinal conditions
Tolerance to artificial simulated gastrointestinal conditions of 21 isolated Bifidobacterium strains was determined and the results were seen in Table 2.The survival rates in artificial simulated gastric juice of all 21 Bifidobacterium strains declined in various degrees.Out of 21 strains, 16 strains exhibited good tolerance to artificial simulated gastric juice, and the survival rate of AHC28 was the highest with 90.40 ± 4.60%,which showed significant difference with the reference strain BB-12 (75.49± 4.60%) (P< 0.05).Subsequently, the survival rates in artificial simulated intestinal juice of 14 strains still remained at a relative high level, and AHC28 also displayed the best tolerance with a 79.61 ± 5.65% survival rate.In general, 14 of the 21 isolated strains exhibited high tolerance to artificial simulated gastrointestinal conditions and AHC2, AHC3, AHC6, AHC9, AHC10, AHC12, AHC15, AHC19, AHC21, AHC24, AHC26, AHC27, AHC28and AHC32 were selected for the succedent experiment.

Adhesionto HT-29 cells
Human colonic carcinoma cell line HT-29 was applied to evaluate the adhesion capacities of 14 Bifidobacterium isolates tointestinal mucosa, and the results were presented in Table 3.The  Results are expressed as the mean ± SD; a-c , different letters along the column represent statistical significance (P< 0.05). https://doi.org/10.1371/journal.pone.0287799.t003 adhesion rates of 14 isolates to HT-29 cells ranged from 2.06 ± 0.21% to 25.45 ± 1.63%, AHC12 and AHC28 showed the highest adhesion rate, but there was no significant difference compared with the control strain BB-12(24.98± 4.38%)(P> 0.05).Six isolates showed lower adhesion ability to HT-29 cell line including AHC2, AHC6, AHC15, AHC19, AHC26, and AHC27 which were not selected to participate in further tests.

Antioxidant capacity
The antioxidant properties of 8 Bifidobacterium isolates including AHC3, AHC9, AHC10, AHC12, AHC21, AHC24, AHC28 and AHC32 were detected by measuring the DPPH, ABTS and superoxide anion radical scavenging activities and the results were observed in

Antibacterial activity
Four Bifidobacterium isolates AHC3, AHC10, AHC24 and AHC28 were tested for their antibacterial activities against 4 pathogenic strains, including E. coli, S. aureus, S. Enterica and S. Sonnei, and the results were indicated in Table 5.All the 4 isolates had bacteriostatic effects on S. aureus.Among them, the antibacterial activity of AHC3 reached to 16.1 ± 2.4 mm, significantly higher than that of the reference strain BB-12 (11.1 ± 3.4) (P<0.05).All the 4 isolates showed bacteriostatic abilities on E. coli except AHC10.Three isolates exhibited inhibitory effects on S. enterica, including AHC3, AHC10 and AHC28.Out of the 4 isolates, only 2 Results are expressed as the mean ± SD; a-d , different letters along the column represent statistical significance (P< 0.05). https://doi.org/10.1371/journal.pone.0287799.t004 isolates displayed inhibitory effects on S. sonnei, and no antibacterial abilities of AHC24 and AHC28 was observed on S. sonnei.Noticeably, among the 4 isolates, only AHC3 possessed antibacterial activities against the 4 tested pathogenic strains which had a broad spectrum of inhibition in our research, it was reasonable to choose AHC3 for the next step of safety test.

Safety of Bifidobacterium strain AHC3
The hemolysis, biogenic amines production and antibiotic sensitivity of isolated Bifidobacterium strain AHC3 were investigated, and the results were presented in Table 6.No hydrolytic circle and color change observed confirmed that AHC3 had absence of hemolytic activity and production of biogenic amines, respectively.Out of 12 commonly used antibiotics, AHC3 was sensitive to 8 kinds of antibiotics, including Gentamicin, Penicillin, Chloroamphenicol, Rifampicin, Erythromycin, Clindamycin, Ciprofloxacin and Ampicillin.On the contrary, it was resistant to Vancomycin, Kanamycin, Streptomycin and Tetracycline.MTT method was employed to determine the cytotoxicity of AHC3 to HT-29 cell line, and the result was shown in Fig 2 .The survival rate of HT-29 cells did not declined significantly, but remained at a high level (93.61 ± 2.61%).Compared with the reference strain BB-12 (94.47 ± 3.76%), no Results are expressed as the mean ± SD; a-b , different letters along the columnrepresent statistical significance (P< 0.05); -, no antibacterial activity detected.

SEM imaging on AHC3
The morphological characteristic of AHC3 strain under scanning electron microscope was seen in Fig 1C and 1D.Under SEM imaging, AHC3 strain was rough on the surface and short rod or ellipsoid.

16S rRNA gene sequencing and identification
The 16S rRNA gene of AHC3 was sequenced and identified.The 16S rRNA gene sequencing results of AHC3 were uploaded to NCBI (the accession No. MN 736535), and BLAST was employed for sequence similarity analysis.The phylogenetic tree constructed of AHC3 was displayed in Fig 3 .The homology of AHC3 and Bifidobacterium breve 16S rRNA gene sequence was up to 100%.Consequently, according to the 16S rRNA gene sequencing and the results of the above physiological and biochemical preliminary identification, AHC3 strain was identified as Bifidobacterium breve and simply named B. breveAHC3.

Pathological evaluation of intestinal tissue in NEC model
Light microscopic images of pathological features of intestinal tissue sections from neonatal SD rats in group A, B, C and D after H&E staining were shown in Fig 4 .After modeling, severe separation of submucosa or lamina propria, disintegration, separation, shedding and loss of intestinal villi glands were observed under light microscope in group C. In contrast, after modeling and B. breve AHC3 gavage in group D, the histopathologic changes were milder than those in group C, and slight edema of submucosa and muscle layer, moderate separation of submucosa and lamina propria, and existence of intestinal villi structure were observed.Intestinal villi in group A and group B were intact, and there was no significant difference between the two groups.

Intestinal histological scoreof intestinal tissue in NEC model
The intestinal histological scores of 4 groups were illustrated in Fig 5A .Among the 4 groups, the intestinal histological score in group C was the highest (3.271±0.914),which was significantly higher than that in group A (0.438±0.260) (P<0.05).Compared with group C, the intestinal histological score of group D significantly declined with the score of 1.438±0.571(P<0.05), while no significant difference was observed between group A and group B (P>0.05).In addition, the incidence of NEC in each group waspresentedin Fig 5B .The incidence rates of NEC in group A, B, C and D were 0% (0/32), 0% (0/32), 81.25% (26/32) and 34.38% (11/32), respectively.Compared with group C, the incidence rate of NEC in group D significantlydecreased (P<0.05).

Evaluation of inflammatory response in the intestinal tissue of NEC model
In order to investigate the effect of B. breve AHC3 on the inflammatory response in NEC model, the inflammatory cytokines in the intestinal homogenate of 4 groups were detected by ELISA, including proinflammatory factor TNF-α and antiinflammatory cytokine IL-10.The results were shown in Table 7.In the 4 groups, the level of TNF-α in group C was the highest, reaching up to 70.968±2.538pg/mL,which was significantly higher than that in group A (P<0.05).After the intervention of B. breve AHC3, the level of TNF-α (41.086±1.134pg/mL) in group D declined,which was significantly lower than that in group C (P< 0.05).Contrary to the results of TNF-α, the level of IL-10 in group D (150.817±10.106pg/mL) was significantly higher than that in group C (79.935±11.143pg/mL)(P< 0.05).No significant difference in the levels of TNF-α and IL-10 was observed between group A and group B.

Evaluation of iNOSexpressionin intestinal tissue of NEC model
The iNOSexpression in intestinal tissues was determined by immunohistochemical staining.
The iNOS gray values in intestinal tissues of the 4 groups were indicated in

Discussion
Tolerance test to artificial gastrointestinal conditions is one of the necessary conditions for screening potential probiotic candidate [54].Sim et al. [55] reported the research on thetolerance to artificial simulated gastrointestinal juices of E. faecium L11 isolated from chicken cecum, and the viability of E. faecium L11 was 66.8 ± 3.3% and 62.8 ± 5.9% respectively after the treatment of artificial simulated gastric juice and intestinal juice, which was lower than the results of our study.Similar to the results of the present work, 4 selected Bifidobacterium strains were cultured in artificial simulated gastric juice at pH 2.0 for 3 h, the survival rates of all the 4 strains exceeded 80%, and the survival rate of B. pseudocatenulatum IMAUFB084 reached to 85.98% [56].Liu et al. [57] concluded that among 6 Bifidobacterium spp.strains, B. breve A03, B. Breve A04 and B. adolescentis L-1 showed better tolerance to artificial gastrointestinal juices, and their survival rates were 87.8%, 89.9% and 89.4% respectively, which was superior to the results of this study.In the present research, the tolerance of individual Bifidobacterium strain to artificial simulated gastrointestinal conditions varied among strains.Compared with the other 7 strains, 14 strains had significantly higher tolerance to artificial gastrointestinal conditions, and the final survival rates exceeded 50%, suggesting that the survival ability of Bifidobacterium stain in artificial simulated gastrointestinal conditions is strainspecific.
In order to investigate the probiotic potential of Bifidobacterium strains, it is necessary to assay the adhesion ability one by one [58].In addition, the HT-29 cell line has been usually employed as a model system simulating the adhesion to human intestinal epithelial cells for in vitro method to evaluate the adhesion ability of probiotics [59,60].It was reported thatB.dentium N8 showed higher adhesion rate to HT-29 cells with 55.64%, indicating that it possessed Results are expressed as the mean ± SD; a-c , different letters along the column represent statistical significance (P< 0.05). https://doi.org/10.1371/journal.pone.0287799.t007 the preeminent adhesion ability [61].Jang et al. [62] used HT-29 cell line to assay the adhesion ability to intestinal mucosa, which showed that B. longum subsp.longumCACC517 strain had the highest adhesion activity to HT-29 cell line (61.7%).Other study also revealed that B.animalis ssp.Lactis UABla-12 showed the adhesion rate of 79-86% to HT-29 cells [63].In our research, 8 of the 14 Bifidobacterium isolates exhibited high adhesion to HT-29 cell line, and the adhesion capabilities of AHC12 and AHC28 were comparable to the reference strain BB-12.The aggregation and hydrophobicity phenotypes may be involved in adhesion, but they are not the unique mechanisms related to adhesion.Additionally, the moonlighting protein transaldolase has been proved to play a part in the adhesion of Bifidobacterium [59,64].Bifidobacterium possesses strain-specific antioxidant property and reduces oxidative damage [65].DPPH, ABTS and superoxide anion radical scavenging, as three frequently-used experimental methods, are currently used to assay the antioxidant property of probiotics [58,66].In the present research, 4 isolates of AHC24, AHC10, AHC3 and AHC28 had respectable DPPH, ABTS and superoxide anion radical scavenging capacities.Our results were consistent with other studies on the antioxidant property of Bifidobacterium.B. longum BL-10 isolated from healthy baby feces by Dong et al. [67], exhibited better antioxidant performance than B. animalissubsp.lactis BB-12.It was illustrated by Yu et al. that the scavenging activities of B. pseudolongum YY-26 on DPPH radical and superoxide anion radical were 97.58% and 43.13%, respectively [68].Kang et al. [69] also confirmed that B. bifidum MG731 showed the highest DPPH radical scavenging activity (90.6%), and B. lactis MG741 displayed the highest ABTS radical scavenging activity (99.5%).The results of the above two studies were better than those of this study.The antioxidant property of Bifidobacterium may be related to a variety of antioxidant compounds, such as soluble proteins and exopolysaccharides [70,71].They may also play a role through specific molecular mechanisms in charge of barrier against the oxidative stress [72].Each antioxidant substance has its own special mechanism of action, resulting in species-and strain-specific antioxidant property [73].
The determination of antimicrobial activity is a necessary step in the screening process of Bifidobacterium [74,75].Aalipanah et al. [37] used disk diffusion method to evaluate the antibacterial property of B. bifidum strain originated from chicken intestinal tract, which had antibacterial property against E. coli.Jang et al. [62] reported that B. longum CACC517 isolated from dog feces showed antibacterial activity against S. enteritidis and E. coli.Cai et al. [76] also revealed that 14 representative Bifidobacterium strains isolated from infant feces in the Ughur population of northwestern China exhibited strong antibacterial activities.Among the 14 strains investigated,B.longum subsp.infantis (BF48-2, BF17-4, BF67-13) and B. bifidum (BF87-11, BF52-1) inhibitedE.coli, S. enterica and S. aureus, with a wide spectrum inhibitory effects.The results of the current work were basically consistent with those of the above studies.Among the 4 isolates, only AHC3 possessed antibacterial activities against the 4 tested pathogenic strains.It was indicated that AHC3 had respectable antibacterial activity.The antibacterial activity of Bifidobacterium may be due to the organic acids such as acetic acid, propionic acid, phenyllactic acid and free fatty acids, and protein compounds including bacteriocin and ablatin produced during the metabolism of Bifidobacterium [77,78].
As the main virulence factor, hemolysis is a special feature of pathogen.The selected Bifidobacterium strains must be absence of hemolytic activity to be safely used in the host body [79].
In the present research, no hemolysis of B. breveAHC3 was observed, which was consistent with the research results of Cizeikiene and Jagelaviciute [65].Biogenic amines are a kind of low molecular mass organic based with aromatic and heterocyclic structures, which is harmful to human health [80].In this study, no biogenic amines production of B. breveAHC3 was observed.It was consistent with the research results of Ku et al. [25] on the biogenic amines production by Bifidobacterium.The isolates may carry antibiotic resistance genes, which could be transmitted to pathogens through horizontal gene transfer, resulting in the emergence of antibiotic resistant pathogens [79].The result of our work was consistent with the studies on antibiotic sensitivity of Bifidobacterium.Choi et al. [81] reported that B. breve IDCC4401 isolated from infant feces showed antibiotic resistance to vancomycin.Zuo et al. [59] found that B. breve IF2-173 and IF2-174 had resistance to Kanamycin and Streptomycin.Kim et al. [79] also observed that B. breve MG729 was resistant to Tetracycline.Bifidobacterium is generally resistant to Gentamicin, Kanamycin and Streptomycin [82].The genetic basis of these drug resistances is chromosome mutation, so they are unlikely to transfer to other bacteria [83].It was reported that a fecal isolate of B. breve NCIM 5671 exhibited no indication of any cytotoxicity, and the viability of HT-29 cells was not affected, with a survival rate of 94.78 ± 2.27% [84].The results of our research were consistent with the above results.It was indicated that B. breveAHC3 had no cytotoxicity to HT-29 cell line.
Although great progress has been made in the treatment of NEC in recent years, there is still no cure for this disease.Studies have shown that probiotics are the most promising intervention to prevent NECat present [85,86].However, differences in the protective capacity of the strains against NEC rat models were found in a recent comparison of some Bifidobacterium and Lactobacillus strains [87].Selection of appropriate probiotic strains seems to be the key to achieving the protective effect on NEC.It was illustrated that administration of live B. breveAHC3 significantly reduced not only the incidence of NEC (from 81.25% to 34.38%) but also the severity of ileal injury in the present research, suggesting that B. breveAHC3might have the potential in the treatment of NEC.Similar to our results, Khailova et al. [13] reported that oral administration of Bifidobacterium could reduce the severity and incidence of NEC.Compared with NEC group, the rats had significantly lower ileal injury and the incidence of NEC in Bifidobacterium intervention group.Underwood et al. [31] also observed that administration of B. longumsubsp.infantishad protective effect on neonatal rat model of NEC, which was characterized by a decrease in NEC incidence and histological score.It was found by Zhou et al. [27] that Bifidobacterium had a protective effect on the intestinal tract of newborn rats suffering from NEC, it could reduce the severity of NEC and intestinal injury.Probiotics added into the intestine may help to reduce the risk of NEC, but the exact mechanism is still unclear.There may be multiple mechanisms to protect the intestine, including stimulating mucosal immune defense response, enhancing the integrity of mucosal barrier, increasing the production of secretory IgA, promoting the development and maturation of intestinal immune function, and diversifying intestinal microbiota, etc. [88].
Inflammation, as an innate immune response, is a basic self-protective response in the human body [89].However, due to the immaturity of intestine and immune system, neonates are prone to excessive inflammation, which may be a key factor in the pathogenesis of NEC [17].A number of different proinflammatory and antiinflammatory factors have been proved to be associated with NEC.In NEC model, some probiotics have been identificated to inhibit the inflammatory reaction to maintain the intestinal barrier function.Xiang et al. [51] found that compared with NEC group, Bifidobacterium could significantly reduce the secretion of TNF-αand alleviate the injury of NEC by inhibiting the release of inflammatory cytokines in Bifidobacterium intervention group.Lueschow et al. [90] reported that B. infantis EVC001 significantly reduced the level of inflammatory cytokines.It was documented that B. fragilis strain ZY-312ZY-312 not only reduced the expression of TNF-α, but also enhanced the expression of IL-10, thus effectively alleviating inflammation and preventing the development of NEC [91].The results of our research were similar to those of the above studies.In this study, after B.bre-veAHC3 intervention, it was indicated that B.breveAHC3 could effectively reduce the level of proinflammatory factor TNF-α and increase the level of antiinflammatory factor IL-10, which had anti-inflammatory potential in the intestinal tract of NEC rats.The mechanism of protective effect on developing intestine from probiotics has not been fully understood.The application of probiotics (such as Bifidobacterium) may inhibit the growth of pathogenic bacteria and reduce the intestinal production of proinflammatory cytokines by reducing the pH value in intestinal cavity and increasing the production of antibacterial substances [92,93].
iNOS is one of the influential enzymes responsible for NEC process [94].iNOSis transcribed and translated under the stimulation of LPS and some cytokines.Once induced, it continuously synthesizes NO.The continuous release of NO may lead to intestinal cellular damage and intestinal barrier failure [22].At the same time, excessive NO activates and amplifies immune and inflammatory reactions, and these proinflammatory factors in turn up-regulate the expression of iNOS, resulting in aggravating epithelial cell damage [95].The upregulation of iNOSsimultaneously inhibits the intrinsic repair mechanism, such as intestinal cell migration and proliferation.The imbalance between epithelial cell damage and repairsresults in the persistent mucosal defects, which further aggravates intestinal injury and enterocyte apoptosis of NEC [96].Application of probiotics reduces the expression of iNOS during NEC, so as to alleviate the injury of NEC.Hunter et al. [97] reported that Lactobacillus decreased intestinal epithelial damage in experimental NEC by reducing iNOS production and maintaining epithelial integrity.Underwood et al. [31] also proved that administration of B. longum subsp.infantis significantly reduced the expression of iNOS.The results of the present research were consistent with the above results.Through the intervention of B.breveAHC3, the gray value of iNOS and expression of iNOS in intestinal tissue of NEC rats was significantly reduced and inhibited, respectively.This may be partially attributed to the reduction of TNF-α level by the intervention ofB.breveAHC3, as the proinflammatory cytokine TNF-αis usually considered as a strong inducer of iNOS expression.In addition, exogenous supplementation of Bifidobacterium contributes to prompting the immune system to play an active role in eliminating oxygen free radicals, inhibiting the production of lipid peroxides, protecting enterocellular organizational structure and maintaining the role of mucosal barrier [98,99].

Conclusions
To sum up, the B. breve AHC3 isolated from the chicken intestines not only exhibited excellent probiotic potential, including high tolerance to artificial simulated gastric conditions, strong adhesion to HT-29 cells, outstanding antioxidant capacity and broad-spectrum inhibition on 4 pathogenic strains, but also possessed reliable safety, such as lack of hemolysis, no biogenic amine production, absence of cytotoxicity, and normal antibiotic sensitivity in the present research.Additionlly, in the neonatal SD rat model of NEC, through reducing the inflammatory reaction in the ileum and inhibiting the expression of iNOS in intestinal tissue cells, B. breve AHC3 had an available protective effect on intestinal injury of NEC.B. breve AHC3 may be a promising candidate strain with probiotic function for the treatment of human NEC.

Table 7 .
Compared with the iNOS gray value in group A (85.730±9.637),after NEC modeling, the iNOS gray value in group C (133.864±23.297)significantlyincreased (P<0.05).After the intervention of B. breve AHC3, the iNOS gray value in group D (95.958±11.221) was significantly lower than that in group C (P<0.05).No significant difference was observed between group A and group B. The light microscopic observation results of immunohistochemical staining were consistent with the iNOS gray value.The iNOS immunostaining of intestinal tissuein group A and B were the lowest (Fig 6A and 6B), and the number of positive particles and staining intensity in group C (Fig 6C) were significantly higher than those in group A and B. However, after B.
breve AHC3intervention, the iNOS expression in intestinal tissue cells in group D was noticeably suppressed (Fig 6D).